Generation of induced Pluripotent Stem Cells from Human Amnion Epithelium

s / Placenta 32 (2011) S326–S340 S334 oligosaccharide chains we performed experiments on human trophoblast cell lines at various oxygen tensions. We reported that oxygen levels comparable to that experienced by first trimester trophoblast in vivo strictly regulated changes in core-1 O-glycan level and we identified trophoblast proliferation as result of increased Oglycans expression. Little is known about the impact of oxygen on protein glycosylation, to our knowledge, this is the first study that identifies the critical contribution of oxygen to O-glycan expression levels. Collectively, our results emphasize that in a oxygen dependent manner glycans may have regulatory effects on first trimester placenta development. doi:10.1016/j.placenta.2011.07.055 GENERATION OF INDUCED PLURIPOTENT STEM CELLS FROM HUMAN AMNION EPITHELIUM R. Gramignoli , M.C. Hansel , F. Marongiu , M. Nagaya , W.L. Blake , A. Soto-Gutierrez , K. Dorko , J.C. Davila , I.J. Fox , S.C. Strom Department of Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA USA; Department of Biomedical Sciences and Technologies, Universita degli Studi di Cagliari, Cagliari – Italy; Genetically Modified Models Center of Emphasis, Pfizer, Groton, CT – USA; d Pfizer, Chesterfield-St. Louis, MO – USA; Departments of Surgery, University of Pittsburgh, Pittsburgh, PA USA Human Amniotic Epithelial (hAE) cells express gene and surface markers typical of Embryonic Stem (ES) cells and have the ability to differentiate into cells from all three germ layers. Both hAE cell and induced pluripotent stem (iPS) cells could be useful stem cell sources for regenerative medicine, devoid of the ethical or religious concerns associated with hES cells. We report the generation of iPS cell lines from primary human AE cells (AE-iPS) following exposure to lentiviral constructs carrying the reprogramming factors Oct4, Sox2, Klf4, c-Myc, Nanog, and Lin28. AE cells were analyzed for alkaline phosphatase activity and mRNA expression levels of stem cell markers (Oct4, Nanog, Sox2) by qRT-PCR and immunocytochemistry. A complete profiling of AE cells preand post-reprogramming was conducted by Flow Cytometry. After reprogramming the percentage of SSEA-3, SSEA-4, TRA1-60 and TRA1-80 and CD 133/2 were dramatically increased. FACS analysis showed higher side scatter in AE, not present in AE-iPS thought to indicate a higher level of cellular complexity and granularity. hAE iPS colonies have themorphological features, surface marker and gene expression profile of fully reprogrammed cells. Teratoma formation has also been confirmed. These studies indicate that iPS lines can be generated from primary human amnion epithelial cells. doi:10.1016/j.placenta.2011.07.056 THE ISOLATION AND CHARACTERISATION OF TERM HUMAN FETALDERIVED AND MATERNAL-DERIVED MESENCHYMAL STROMAL CELLS C. Heazlewood , G. Brooke , N. Fisk , K. Atkinson a,b Mater Medical Research Institute, Brisbane, Australia; University of Queensland, Brisbane, Australia; c University of Queensland Centre for Clinical Research, Brisbane, Australia The placenta is rich in mesenchymal stromal cells (MSC) and consists of both fetal (amnion and chorion membranes) and maternal (decidua) tissues. Fetal-derived MSC may have different biological properties from maternal MSC, given the differences in age at the time of their development, and such differences may have implications in the potential use of MSC as therapeutic agents. Therefore, we focused on isolating and characterising fetal and maternal MSC of the human placenta. Male gender babies from elective term caesarean sections were collected, the amniotic and chorionic membranes were mechanically separated and the decidua tissue removed. The tissues were digested and the mononuclear cell fraction collected. The cells were either plated in culture or stained with primitive cell surface marker antibodies to determine if primitive cell populations could be isolated. Genotyping was also performed on cells that had been in culture to determine source of origin. Expanded cells were characterised according to cell surface phenotype and ability to differentiate into mesodermal lineages. Cells from the amnion, chorion and decidua had an adherent, fibroblast-like morphology and exhibited typical MSC characteristics such as cell phenotype and differentiation ability, although there was little differentiation into adipocytes. Very little cell surface expression of the primitive stem cell markers Tra1-60, Tra1-81 and SSEA-4 was found in freshly sorted cells. Genotyping showed the amnion cells were predominately fetal in origin and chorion and decidua cells were predominately maternal in origin. It remains to be determined if fetal MSC show greater therapeutic ability than maternal MSC. doi:10.1016/j.placenta.2011.07.057 AMNIONIC MESENCHYMAL STROMAL CELLS (AMSC) SHOW A MESENCHYMAL-EPITHELIAL PHENOTYPE AND ADOPT ENDOTHELIAL CHARACTERISTICS UNDER ANGIOGENIC CONDITIONS J. Konig , B. Huppertz , G. Dohr , O. Parolini , I. Lang a a Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Austria; b Centro di Ricerca E. Menni, Fondazione Poliambulanza, Istituto Ospedaliero, Brescia, Italy Even though AMSC play an important role in stem/progenitor cell research, their characterization is mostly limited to immunodetection by flow cytometry. Therefore, we performed immunohistochemical studies on cultured cells and term placental cryosections. Furthermore, we cultured the cells under angiogenic conditions to investigate their endothelial differentiation potential and examined the interaction with endothelial cells (EC). AMSC express the common mesenchymal stem cell markers CD73, CD105 and CD90 and seem to adopt a mesenchymal-epithelial phenotype during culture: Shortly after isolation (1d), 92.1% ( 5.6%) of the cells only express the mesenchymal marker vimentin, 1.3% ( 1.4%) solely stain for the epithelial marker cytokeratin-7 and 5.7% ( 3.4%) co-express these markers. After 5d, the double positive cells increase to 9.9% ( 3.4%), while exclusive expression of cytokeratin or vimentin remains about the same (1.2% 1.3% and 88.8% 3.8%, respectively). After passage 1, all cells are vimentin-positive, while 50-80% co-express cytokeratin-7 and vimentin. AMSC express vascular endothelial growth factor receptor-2 which can also be found on endothelial precursor cells. Angiogenic stimulation with endothelial growth medium containing VEGF enhances their proliferation potential and viability. They change their fibroblast-like morphology towards an endothelial, cobblestone-like phenotype. Even though they do not express the mature EC markers vWF and CD144, they take up DiIAcLDL, a characteristic of EC. Induced AMSC form more stable and widespread networks than EC in a network formation (matrigel) assay. In coculture, they adhere to EC and stabilize their networks. These data indicate angiogenic properties of AMSC which might be of therapeutic use in vascular biology. doi:10.1016/j.placenta.2011.07.058 IMMUNOREGULATORY PROPERTIES OF HUMAN AMNIOTIC MESENCHYMAL STROMAL CELLS: A COMPARISON TO HUMAN ADIPOSE DERIVED STEM CELLS B. Kronsteiner , S. Wolbank , A. Peterbauer-Scherb , M. Van Griensven , H. Redl , C. Gabriel a,c a Red Cross Blood Transfusion Service of Upper Austria, Linz, Austria; b Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Center, Linz/Vienna, Austria; c Austrian Cluster for Tissue Regeneration, Vienna, Austria